1. Field of the Invention
The invention relates to a process for preparing non-proteinogenic L-amino acids by means of enzymic biotransformation.
2. The Prior Art
Non-proteinogenic amino acids are amino acids which are not used in nature as building blocks for protein biosynthesis and are thereby to be clearly demarcated from the 20 proteinogenic amino acids. Within the meaning of the present invention, the very rare amino acid L-selenocysteine, which does indeed occur in proteins, is classified among the non-proteinogenic amino acids.
Non-proteinogenic amino acids constitute interesting compounds, for example for producing pharmaceutical and agricultural active compounds. They are able, as an active compound or as part of an active compound, to imitate the structure of natural amino acids in a type of molecular mimicry and thereby modulate the natural reaction, for example in the case of receptor interactions. Furthermore, as chiral compounds, they can, quite generally, serve as synthetic building blocks within the context of the xe2x80x9cchiral poolxe2x80x9d.
Present methods for preparing non-proteinogenic amino acids in enantiomerically pure form are for the most part based on syntheses which are elaborate and which, furthermore, usually only permit access to one particular compound. Only a few methods enable different compounds to be prepared by simply replacing a starting compound. In most cases, the syntheses are chemical syntheses which, for their part, usually already proceed from chiral building blocks. Other methods combine the chemical synthesis of racemates with a racemate resolution which is frequently carried out enzymically.
In addition, some enzymic methods which make use of prochiral compounds and which enable non-proteinogenic amino acids to be synthesized stereoselectively have also been described. Thus, it is possible to employ transaminases to prepare various non-proteinogenic amino acids from xcex1-keto acids using L-glutamic acid as the amino donor (Taylor et al. 1998, TIBTECH 16: 412-418). Another example is the synthesis of L-tert-leucine using leucine dehydrogenase (Drauz 1997, Chimia 51: 310-314).
The patent application DE 10046934 (registered on 21.09.2000 by the same applicant) describes a particularly simple method for preparing non-proteinogenic amino acids by means of direct fermentation of microorganisms. This method uses organisms whose cysteine metabolism is deregulated and which therefore supply a high level of O-acetyl-L-serine. In cysteine metabolism, this compound serves as a biosynthetic precursor of L-cysteine. The latter is formed by substituting the acetate group at the xcex2 position with a thiol radical. This reaction, which is termed xcex2 substitution, is catalyzed by enzymes of the O-acetyl-L-serine sulfhydrylase class [EC 4.2.99.8]. When nucleophilic substances belonging to particular compound classes (thiols, azoles and/or isoxazolinones) are fed in during the fermentation, these compounds enter into xcex2 substitution, thereby achieving the production of non-proteinogenic L-amino acids. The structure of the respective radicals of the amino acids which are prepared are thus dictated by the nucleophilic compound which is supplied.
A problem with this method is that the nucleophilic compounds which are fed in should not be metered in at too high a rate since, otherwise, the compound itself, or the resulting amino acid, can elicit toxic effects on the metabolism of the microorganisms. This applies, in particular, to many thiol compounds since, as redox-active substances, they possess toxicity at higher concentrations. Furthermore, it is very problematical to use thiol compounds in fermentative methods because, when the fermenter is intensively aerated, they tend toward oxidation and, without mechanical provisions, cause a significant degree of obnoxious odor. Because of their high toxicity, it is not possible, either, to feed in azide or cyanide, which are known to enter into xcex2 substitution when O-acetyl-L-serine sulfhydrylases are used (Flint et al., 1996, J. Biol. Chem. 271: 16053-16067).
It is an object of the present invention to provide a process for preparing non-proteinogenic L-amino acids, which process makes it possible to use nucleophilic compounds, in particular toxic compounds as well, which are metered in at a high rate.
This object is achieved according to the present invention by means of an enzymic biotransformation method in which O-acetyl-L-serine is reacted with a nucleophilic compound, while using an O-acetyl-L-serine sulfhydrylase as catalyst, to give a non-proteinogenic L-amino acid, which comprises the process being carried out at a pH in the range between pH 5.0 and 7.4.
This process makes it possible to synthesize a large number of non-proteinogenic, enantiomerically pure L-amino acids, some of which are novel, on an industrial scale.
O-Acetyl-L-serine sulfhydrylases are known. They have so far been isolated from a very wide variety of plants and microorganisms. Those which have been investigated to the greatest extent are the corresponding bacterial enzymes isolated from Salmonella typhimurium. In this organism, there are two O-acetyl-L-serine sulfhydrylase enzymes, which are designated OASS-A and OASS-B, respectively. The pertinent genes are likewise known and are termed cysK and cysM, respectively. Although the two enzymes possess very similar reaction mechanisms, they only exhibit 45% identity on the basis of their amino acid sequences.
OASS-B (CysM) has been reported to be able, in contrast to OASS-A (CysK), to catalyze a reaction of O-acetyl-L-serine with thiosulfate to give S-sulfocysteine. This reaction plays an important role in the growth of the bacteria when thiosulfate is the sole sulfur source.
Furthermore, comparisons of the sequences of O-acetyl-L-serine genes from various organisms indicate that there are two phylogenetic groups (Kitabatake et al., 2000, J. Bacteriol. 182: 143-145). Salmonella typhimurium CysK and Escherichia coli CysK form a large group together with the O-acetyl-L-serine sulfhydrylases from other Eubacteria, from methanogenic Archaea and from plants. By contrast, Salmonella typhimurium CysM and Escherichia coli CysM are located in a very small family together with the O-acetyl-L-serine sulfhydrylases from hyperthermophilic Archaea (e.g. Pyrococcus, Sulfolobus and Thermoplasma).
Within the meaning of the present invention, O-acetyl-L-serine sulfhydrylases are distinguished by the fact that they are able to catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. Both CysM-related and CysK-related enzymes are therefore O-acetyl-L-serine sulfhydrylases within the meaning of the present invention.
Although a few publications show that O-acetyl-L-serine sulfhydrylases from the CysK group exhibit a relatively broad substrate spectrum (Ikegami and Murakoshi, 1994, Phytochemistry 35: 1089-1104; Flint et al., 1996, J. Biol. Chem. 271:16053-16067), the possibility of using O-acetyl-L-serine sulfhydrylases industrially for producing non-proteinogenic amino acids has not previously been considered.
The crucial reason which has previously stood in the way of using O-acetyl-L-serine sulfhydrylases to implement the enzymic preparation of non-proteinogenic amino acids industrially lies in the fact that O-acetyl-L-serine is unstable precisely in the pH range which corresponds to the activity range of the O-acetyl-L-serine sulfhydrylases.
O-Acetyl-L-serine isomerizes to N-acetyl-L-serine in dependence on the pH. The reaction is irreversible and, for example, at a pH of 7.6 extremely rapid, with rates of 1%xc3x97minxe2x88x921. The rate of reaction falls as the pH is lowered such that the compound is stable at pH 4.0, for example. The mechanism of the reaction is based on an intramolecular, nucleophilic attack of the deprotonated amino group on the carbonyl carbon of the acyl radical (Tai et al. 1995, Biochemistry 34: 12311-12322).
By contrast, the pH optimum of O-acetyl-L-serine sulfhydrylases lies in the region of pH 8.0 (Ikegami and Murakoshi, 1994, Phytochemistry 35: 1089-1104; Tai et al. 1995, Biochemistry 34: 12311-12322) and consequently in a region which is very unfavorable with regard to the isomerization of O-acetyl-L-serine.
For this reason, Ikegami and Murakoshi propose a biomimetic synthesis of non-proteinogenic amino acids using O-acetyl-L-serine, a nucleophilic compound, pyridoxal phosphate and metal ions (preferably Ga2+). This reaction can take place in a pH range of from 3.5 to 5.5 and consequently ensures the stability of O-acetyl-L-serine. However, with maximum yields of  less than 45%, the efficiency of this method is not very high. A major disadvantage as compared with the enzymic synthesis is the lack of enantioselectivity.
Another object, which is achieved by the process according to the invention, was, therefore, to provide an enzymic process for preparing enantiomerically pure, non-proteinogenic amino acids which, despite the incompatibility of the O-acetyl-L-serine stability and the enzyme activity optimum, ensures an efficient enzymic turnover ( greater than  greater than 45%) in association with a low level of isomerization.
The object was achieved by the process according to the invention since this process is preferably distinguished by the fact that
the reaction is carried out below the pH optimum of the O-acetyl-L-serine sulfhydrylases, and
the enzyme is used in sufficiently high dosage.
The preferred pH range for preparing non-proteinogenic amino acids within the meaning of the present invention is the pH range between pH 5.0 and 7.4.
The pH range is particularly preferably between pH 6.0 and 7.1.
The pH range is especially preferably between pH 6.0 and 6.99.
The pH is preferably kept constant by regulating it actively in order to counteract the stoichiometric formation of acetic acid as well. The active regulation of the pH is preferably accomplished by means of a measuring and control unit which, when the pH deviates from the required value, resets it by metering in an alkali or acid.
Contrary to the approach adopted in accordance with the invention, the reactions using O-acetyl-L-serine sulfhydrylases for synthesizing non-proteinogenic amino acids which have been previously described in the literature have been carried out in the region of the pH optimum of the O-acetyl-L-serine sulfhydrylases without any active regulation of the pH and solely on an analytical scale. Furthermore, they have without exception used enzymes belonging to the phylogenetic group to which the CysK enzymes belong.
The invention consequently also relates to a process for preparing a non-proteinogenic L-amino acid in which O-acetyl-L-serine is reacted with a nucleophilic compound, while using an O-Acetyl-L-serine sulfhydrylase as catalyst, to give a non-proteinogenic L-amino acid, which comprises using CysM as the O-acetyl-L-serine sulfhydrylase.
An adequately high dosage of the enzyme ensures that sufficient turnover takes place even outside the pH optimum of the enzyme reaction. Such an enzyme concentration is then preferably achieved for the process when the volume activity of the O-acetyl-L-serine sulfhydrylase, Acys, is at least 2 units/ml in the mixture. The activity is particularly preferably 2-200 units/ml. The activity is determined using the test described in example 3.
During the course of the present invention, it was possible to observe that O-acetyl-L-serine sulfhydrylases from the phylogenetic group to which the CysM enzymes belong are also outstandingly good enzymes for preparing non-proteinogenic amino acids. Surprisingly, the spectrum of the nucleophilic compounds which are suitable for use as the substrate is even broader than that of the CysK enzymes.
In a particularly preferred embodiment of the invention, the enzyme reaction is conducted as a continuous process. In this case, O-acetyl-L-serine, O-acetyl-L-serine sulfhydrylases and nucleophilic compound are metered in constantly and, at the same time, a solution containing the non-proteinogenic L-amino acid (product solution) is withdrawn from the mixture. The latter preferably takes place such that the volume in the reaction mixture remains the same. The particular advantage of this procedure is that a steady state is set up such that there is a constantly low concentration of O-acetyl-L-serine in the mixture and the isomerization is consequently decreased. Preferably, the concentration of O-acetyl-L-serine in the mixture is adjusted to  less than 1.0 g/l. This value can be controlled by varying the mean dwell time of the solution in the reaction mixture. The reservoir of O-acetyl-L-serine for the continuous reaction is preferably maintained at an acidic pH, preferably at pH 4-5, in order to ensure sufficient stability.
O-Acetyl-L-serine has previously only been available from chemical synthesis, by the acetylation of L-serine, and has been expensive due to the high prices for L-serine. The application DE 10107002, which belongs to the same applicant and which was filed on Feb. 15, 2001, describes a fermentative method for preparing O-acetyl-L-serine. While this application makes available a cost-advantageous production system, there are difficulties with isolating the product from the fermenter broth due to the instability of the O-acetyl-L-serine.
An advantage of the present invention is that an O-Acetyl-L-serine-containing fermenter broth, as is obtained, for example, from a fermentation which is carried out as described in DE 10107002, can be used directly as the O-acetyl-L-serine source in the process according to the invention. This approach is particularly economical and avoids the isolation of an unstable compound.
O-Acetyl-L-serine sulfhydrylases for synthesizing the non-proteinogenic amino acids are preferably prepared using customary recombinant DNA techniques with which the skilled person is familiar.
For this, a gene which encodes an O-Acetyl-L-serine sulfhydrylase is cloned into a suitable vector and a suitable host strain is subsequently transformed. Any microorganism which is accessible to recombinant DNA techniques and which is suitable for fermentatively preparing recombinant proteins is suitable for use as the host strain.
Escherichia coli is a preferred microorganism for preparing O-acetyl-L-serine sulfhydrylases.
In principle, it is possible for the recombinant O-acetyl-L-serine sulfhydrylase gene to be integrated into the chromosome or else to be used on a self-replicating plasmid vector.
In the cloning, preference is given to using vectors which already contain genetic elements (e.g. regulatable promoters, terminators) which enable the O-acetyl-L-serine sulfhydrylase gene to be expressed in a controlled and strongly inducible manner. Particular preference is given to plasmid vectors which are present in high copy number, such as the Escherichia coli vectors pUC18, pBR322, pACYC184 and their derivatives. Examples of suitable strongly inducible promoters are the lac, tac, trc, lambda PL, ara and tet promoters.
O-Acetyl-L-serine sulfhydrylases are produced, for example, by cultivating a recombinant microorganism strain by means of fermentation. In this connection, use is made of propagating methods which are known to a skilled person, with the methodological parameters having to be adapted to the given microorganism strain. Both complete media and minimal media can be used as nutrient media. It is possible to use either a batch process or a fed-batch process. When inducible promoter systems are used, expression of the O-acetyl-L-serine sulfhydrylase gene is switched on at a suitable point in time by adding an appropriate inducer. Following an adequate production phase, the O-acetyl-L-serine sulfhydrylase-containing cells are harvested using known methods (e.g. centrifugation).
The O-acetyl-L-serine sulfhydrylase enzyme which is prepared in this way can be isolated using customary methods of protein purification. In this connection, it is possible to use both classical methods (e.g. precipitation, ion exchange chromatography, hydrophobic interaction chromatography and isoelectric focussing) and modern affinity chromatography employing xe2x80x9caffinity tagsxe2x80x9d. Sequences which encode these affinity tags can be fused to the coding region when the gene is cloned, thereby giving rise to fusion proteins which contain the corresponding affinity tag. These proteins are then isolated in a one-step purification. An example of a suitable combination of affinity tag and affinity purification is the Strep-Tag and Streptavidin affinity chromatography, which combination can be purchased from IBA, Gxc3x6ttingen, Germany.
It is possible to use an O-Acetyl-L-serine sulfhydrylase in purified form in the process according to the invention. However, in addition to the reaction in solution, it is also possible to immobilize the enzyme on a support. Appropriate methods belong to the state of the art.
However, it is not absolutely necessary to isolate the O-acetyl-L-serine sulfhydrylase for preparing non-proteinogenic amino acids. It is also possible to employ microorganism cells, which possess O-acetyl-L-serine sulfhydrylase activity, directly in the process according to the invention. Examples of such microorganism strains are the Escherichia coli strains DH5xcex1/pFL145 and BLR21(DE3)/pLE4. They are described in examples 1 and 2 and have been deposited in the Deutsche Sammlung fxc3xcr Mikroorganismen und Zellkulturen [German collection of microorganisms and cell cultures], DSMZ, in Braunschweig under the Nos. DSM 14088 and 14089, respectively.
In this variant of the process according to the invention, O-acetyl-L-serine is biotransformed into a non-proteinogenic L-amino acid using resting cells. In this connection, the penetration of O-acetyl-L-serine and a nucleophilic compound into the cells is just as much ensured as the release of the reaction product, i.e. the non-proteinogenic L-amino acid, by the cells.
If desired, transfer of material between the interior of the cell and the reaction medium can be increased by treating the cells with substances which cause the cells to become permeabilized. These substances, for example chloroform or toluene, and their use are known to the skilled person.
In a preferred embodiment of the process, a non-proteinogenic amino acid is formed by the reaction of O-acetyl-L-serine with a nucleophilic compound, while using an O-Acetyl-L-serine sulfhydrylase as catalyst, with the O-acetyl-L-serine sulfhydrylase being present intracellularly in a microorganism.
Particular preference is given to preparing non-proteinogenic L-amino acids by reacting fermentatively obtained, non-purified O-acetyl-L-serine with a nucleophilic compound, while using an O-Acetyl-L-serine sulfhydrylase as catalyst, with the O-acetyl-L-serine sulfhydrylase being present intracellularly in a microorganism.
The nucleophilic compounds which are used in the process according to the invention for the xcex2 substitution which is catalyzed by O-acetylserine sulfhydrylases are preferably compounds which contain a radical which is selected from the group consisting of 
Particular preference is given to adding, to the reaction mixture, a nucleophilic compound which is selected from the group consisting of the following compounds:
thiosulfates
thiols of the general formula (1):
Hxe2x80x94Sxe2x80x94R1xe2x80x83xe2x80x83(1) 
where R1 is a monovalent substituted or unsubstituted alkyl, alkoxy, aryl or heteroaryl radical having from 1 to 15 C atoms;
selenides
selenols of the general formula (2)
Hxe2x80x94Sexe2x80x94R1xe2x80x83xe2x80x83(2) 
where R1 has the meaning given for formula (1),
azides
cyanides
azoles of the general formula (3) or (4): 
where X and Y are identical or different and are CR4 or N, and R4 is xe2x80x94H, xe2x80x94COOH, xe2x80x94OH, xe2x80x94NH2, xe2x80x94NO2, xe2x80x94SH, xe2x80x94SO3xe2x88x92, xe2x80x94F, xe2x80x94Cl, xe2x80x94Br, xe2x80x94I, C1-C5-alkylcarbonyl- and also their esters, amides or salts, or R1, and R1 has the meaning given for formula (1), and
where R2 and R3 are identical or different and are R4 or where C1 and C2 in formula (4) are linked, in place of the substituents R2 and R3, by means of a bridge [xe2x80x94CR5R6xe2x80x94]a, where a is 1, 2, 3 or 4, to form a ring, where
R5 and R6 are identical or different and are R4, and one or more non-adjacent groups [xe2x80x94CR5R6xe2x80x94] can be replaced with oxygen, sulfur or an imino radical which is optionally substituted by C1-C5-alkyl-, and
two adjacent groups [xe2x80x94CR5R6xe2x80x94] can be replaced with a group [xe2x80x94CR5xe2x95x90CR6xe2x80x94] or with a group [xe2x80x94CR5xe2x95x90Nxe2x80x94],
isoxazolinones of the general formula (5) or (6): 
where X, R1, R2 and R3 have the meaning which has already been given, and
where C1 and C2 in formula (6) can be linked, in place of the substituents R2 and R3, by means of a bridge as defined for formula (4) to form a ring.
Examples of thiosulfates are sodium thiosulfate, potassium thiosulfate and ammonium thiosulfate.
Examples of thiols are compounds selected from the group consisting of 2-mercaptoethanol, 3-mercaptopropanol, 3-mercaptopropionic acid, 3-mercapto-1-propanesulfonic acid, mercaptoethanesulfonic acid, 2-mercaptoethylamine, thioglycolic acid, thiolactic acid, thioacetic acid, mercaptosuccinic acid, mercaptopyruvic acid, dithiothreitol, dithioerythritol, 1-thioglycerol, thiophenol, 4-fluorothiophenol, 4-chlorothiophenol, 4-mercaptophenol, p-thiocresol, 5-thio-2-nitrobenzoic acid, 2-mercaptothiazole, 2-mercaptothiazoline, 2-mercaptoimidazole, 3-mercapto-1,2,4-triazole, 2-thiophenethiol, 2-mercaptopyridine, 2-mercaptopyrimidine, 2-thiocytosine, 2-mercaptonicotinic acid, 2-mercapto-1-methylimidazole, 2-mercaptobenzothiazole, 2-mercaptobenzoxazole and 6-mercaptopurine.
Examples of selenols are compounds selected from the group consisting of methylselenol, ethylselenol, propylselenol and phenylselenol.
Examples of azides are sodium azide, potassium azide and ammonium azide.
Examples of cyanides are potassium cyanide, sodium cyanide and ammonium cyanide.
Examples of azoles are compounds selected from the group consisting of 1,2-pyrazole, 3-methylpyrazole, 4-methylpyrazole, 3,5-dimethylpyrazole, 3-aminopyrazole, 4-aminopyrazole, pyrazole-4-carboxylic acid, pyrazole-3,5-dicarboxylic acid, 1,2,3-triazole, 1,2,4-triazole, 3-amino-1,2,4-triazole, 1,2,3,4-tetrazole, indazole, indazole-3-carboxylic acid, indazole-5-carboxylic acid, 5-aminoindazole, benzotriazole, benzotriazole-5-carboxylic acid, 5-aminobenzotriazole, aminopyrazolopyrimidine, 8-azaguanine and 8-azaadenine.
Examples of isoxazolinones are compounds selected from the group consisting of isoxazolin-5-one, 3-methylisoxazolin-5-one, 4-methylisoxazolin-5-one, 4,5-dimethylisoxazolin-2-one and 1,2,4-oxadiazolidine-3,5-dione.
The concentration of the nucleophilic compound in the mixture is preferably selected such that the compound is present in a concentration which is equimolar with that of the O-acetyl-L-serine.
The reaction temperature is preferably selected to be between 5xc2x0 C. and 70xc2x0 C. The temperature range which is particularly preferred is between 20xc2x0 C. and 40xc2x0 C.
Water is preferably used as the solvent for the reaction.
The reaction products which are formed are preferably L-amino acids of the general formula (7) in the L configuration 
where Z is a monovalent radical selected from the formulae (8) to (19) 
and the esters, ethers or salts thereof,
and R1, R2, R3, R4, X and Y have the meaning which has already been given for the formulae (1) to (6).
Preference is given to isolating a soluble non-proteinogenic L-amino acid from the culture supernatant after the process according to the invention has been terminated and after the preferably aqueous solution has been separated, using known methods, into biomass and a culture supernatant. Such methods for isolating amino acids are likewise known to the skilled person. They comprise, for example, filtration, centrifugation, extraction, adsorption, ion exchange chromatography, precipitation and crystallization. In the case of a difficultly soluble non-proteinogenic amino acid, preference is given to carrying out a grading centrifugation, as is known to the skilled person, with the biomass if at all possible remaining in the centrifugation supernatant. The product which has been separated off is preferably dissolved and reprecipitated using standard methods.